1. Field of the Invention
The present invention relates to a molecular recognition probe that expresses electrochemical activity upon recognition of a target molecule, particularly a molecular recognition probe that sequence-specifically expresses electrochemical activity upon recognition of a target nucleic acid, as well as a molecular recognition sensor and electrochemical detection method based on the same.
2. Description of the Related Art
To detect a specific molecule in a solution where multiple molecules coexist, some physical or chemical perturbation is given by using as an indicator a physical/chemical characteristic unique to this molecule, and the obtained signal change is used to identify the molecule.
If the target molecule has no unique characteristic, a molecular group and the like having a characteristic is used to label the molecule and the target molecule is detected by using this label as an indicator. If labeling the target molecule is difficult, in many cases a target recognition reagent that can specifically recognize the target molecule is used, and a characteristic is given to the target recognition reagent to detect the target molecule. Particularly when there are many measuring targets, the latter method is often exploited.
There are growing needs in recent years for high-throughput screening of biomaterials such as nucleic acids, proteins, peptides, and comprehensive bioactivity analyses such as genome analysis, proteome analysis, and metabolome analysis. New detection methods to replace conventional labeling methods are being desired as the scales of measuring targets continue to increase.
Currently when detecting a nucleic acid in genome analysis, for example, the nucleic acid is directly labeled with a fluorescent moiety to be measured (=target nucleic acid) and the target nucleic acid is sequence-specifically hybridized with another nucleic acid having a sequence complementary thereto and immobilized on a chip or substrate (=probe nucleic acid), after which fluorescence is directly detected to identify the target nucleic acid.
If many types of nucleic acids are measured, however, performing this labeling process for all nucleic acids is very cumbersome and label-free nucleic acid detection methods are desired. Such a situation is the same in proteome analysis and metabolome analysis where many proteins and peptides must be handled.
For example, the molecular beacon method is one of the most frequently used methods among label-free nucleic acid detecting methods. As shown in FIG. 6, in this method, both ends of the probe nucleic acid are modified, respectively, with a fluorescent group (F) and a quenching group (Q) that quenches fluorescence from the fluorescent group. When the probe nucleic acid is not hybridized with the target nucleic acid, a hairpin structure is formed where the quenching group is positioned near the fluorescent group to suppress emission of fluorescence. Only when the probe nucleic acid is hybridized with the target nucleic acid, then the fluorescent group is separated from the quenching group and emits fluorescence, thereby indicating the existence of the target nucleic acid (Patent Literature 1, Non-patent Literature 1).
However, while such detection methods based on fluorescence spectroscopy achieve detection with high sensitivity, it also requires a large, expensive measuring equipment consuming a lot of energy.
The inventors for the present invention had been developing simple methods to replace the fluorescence spectroscopy method. In this process, the inventors demonstrated that electrochemical methods would permit simple detection because the detection is possible using a small, inexpensive apparatus consuming less energy. Particularly, needs for simple diagnosis tools are lately increasing further; toxicogenomics, pharmacogenomics, and other technologies, which are diagnosis technologies based on deviations from normalcy in the workings of genes and proteins in a living body and provide patients with pathological prediction and choices for treatment, are seeing an expansion of their application fields from laboratory-level research to clinical diagnosis.
For example, as a label-free electrochemical molecular recognition method, an ion channel sensor was developed by the inventors (Patent Literatures 2, 3, Non-patent Literature 2). This method was developed by focusing on the function of channel protein present in biomembranes to control the flow of a large amount of ions inside and outside of the biomembranes as this protein binds with a small amount of ligand, and specifically this sensor detects the target substance based on molecular recognition and consequent signal amplification. A molecule that can selectively bind to the target substance is fixed on the electrode surface as a receptor and an electrochemically active species (marker) is dissolved in the measurement solution. As the target substance binds to the receptor and condenses at the electrode surface, electron transfer reaction of the marker is facilitated or suppressed at the electrode surface. By putting in place such mechanism, presence of a small amount of the target substance can be detected as a flow of a large mount of electrons. The biggest advantage of this principle is that the target substance can be detected electrochemically even when the target substance itself has no electrochemical activity (=even though the target substance is not labeled). The inventors successfully detected, with high sensitivity, many different molecules, ions, sugar chains, peptides and nucleic acids.
The inventors then advanced this principle one step further and developed a receptor having electrochemical activity, consequently proposing an even simpler method that not only eliminates the need to label the target substance, but also eliminates the need to add a marker (Non-patent Literature 3). Take a receptor for nucleic acid detection (probe nucleic acid), for example. One end of this probe nucleic acid is modified with ferrocene or other electrochemically active group, while the other end is fixed to the electrode surface. It is known that nucleic acids have a very flexible structure in single-stranded form, but assume a rigid structure in double-stranded form. Paying attention to this nature of nucleic acids, the inventors found that sequence-specific detection of the target nucleic acid is possible from a decrease in the observed current, because ferrocene at the end of the probe nucleic acid changes its state, upon hybridization, from one where electron transfer reaction occurs freely at the electrode surface, to one where such reaction is difficult. Detection of various target substances such as proteins, molecules, ions and other types of chemical species using similar methods has also been attempted (Non-patent Literatures 4 to 7).
However, such detection method using a receptor with an electrochemically active species appended at its end must depend on control of electron transfer reaction whose principle is in turn dependent on the distance from the electrode surface, and therefore the mechanism becomes such that a molecular recognition event is notified by a decrease in electron transfer reaction. Almost all probe nucleic acids based on an electrochemical detection principle adopt such mechanism. In general, a system whose signal decreases upon molecular recognition (“signal-off” type) is associated with low detection sensitivity. In contrast, a system whose signal increases upon molecular recognition (“signal-on” type) can expect high detection sensitivity. To develop an analysis method that provides the simplicity of an electrochemical method while eliminating the needs for both labeling and marker, it is necessary to develop a “signal-on” type method. However, no such method has been developed to date.